To elevate chemo-resistance of human cancer cells is a major
obstacle in the treatment and management of malignant cancers. Diallyl
sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS)
are presented in the Alliaceae family particularly in garlic. Although
DAS, DADS and DATS have been shown to exhibit anticancer activities,
there is little information on effects of these compounds on drug
resistant genes in human colon cancer cells in vitro and in vivo.
Herein, we are the first to show that DAS, DADS and DATS at 25 [micro]M
for 24-h and 48-h incubations promoted expression of drug resistant
genes in colo 205 human colon cancer cells. In vitro experiments
indicated that DATS promoted gene expression of multidrug resistant I
(Mdr1) (p<0.05), and DAS and DADS promoted MRP3 gene expression and
DATS alone stimulated gene expression of multidrug resistance-associated
protein-1 (MRPl) (p < 0.05) in colo 205 cells. In vivo studies
demonstrated that DADS and DATS induced Mdr1 and MRP1 gene expression
(p<0.05). DADS promoted MRP3 gene expression (p < 0.05) as well as
DADS and DATS increased MRP4 and MRP6 gene expression (p < 0.05) in
the colo 205 xenograft mice. Based on our in vitro and in vivo results,
diallyl polysulfides (DAS, DADS and DATS) affected the gene expression
of the multidrug resistance in colo 205 human colon cancer cells in
vitro and in vivo.

Drug resistance is a major impediment in the treatment of cancer
(Harnett et al. 1987; Liem et al. 2002). Multiple cytotoxic drugs with
diverse mechanisms of action have not been effective as cancer cells
developed resistance simultaneously to different anticancer drugs.
Advances in understanding the mechanisms of multidrug resistance (MDR)
and associated multidrug-resistant genes in tumor cells or tissues
models are essential to improve cancer therapy (Chao et al. 1991; Fan et
al. 2004; Perez-Tomas 2006). It is reported that MDR of mammalian cancer
cells exhibited that overexpression of permeability-glycoprotems (P-gps)
including a family of mdr genes in membrane and alterations of
ATP-dependent drug efflux and intracellular drug accumulation (Callen et
al. 1987; Fan et al. 2004). Therefore, the novel investigations for
exploring their functions of cancer drug-resistance mechanism are vital
during cancer treatment.

Diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl
trisulfide (DATS) are the sulfur-containing compounds found in the
Alliaceae family such as garlic. These three compounds are thought to be
only part responsible for health-promoting effects such as
antimicrobial, hypolipidemic, antithrombotic, and antitumor activities
(Augusti 1996; Block et al. 2001: Milner 2001; Yin and Tsao 1999).
Epidemiologic studies and laboratory experiments have demonstrated that
garlic has bioactivity and anticancer effects (Bianchini and Vainio
2001; Fleischauer and Arab 2001; Singh and Shukla 1998). It is reported
that DAS, DADS and DATS are agonist of both transient receptor potential
cation channel, subfamily A, member 1 (TRPA1) and transient receptor
potential cation channel, subfamily V, member 1 (TRPV1) but with high
affinity for TRPA1 activity (Koizumi et al. 2009).

DAS enhanced antioxidants and suppresses inflammatory cytokines
through the activation of Nrf2 and it was protective against oxidative
stress induced by gentamicin in Wistar rats (Kalayarasan et al. 2009).
DAS reduced INH-induced toxicity by stabilizing the cellular GSH content
from oxidative injury in rat liver cells (Zhai et al. 2008). DAS exerted
a protective role on liver function and tissue integrity in the face of
enhanced tumorigenesis caused by N-nitrosodiethylamine, as well as
improving cancer-cell sensitivity to chemotherapy (Ibrahim and Nassar
2008).

DAS, DADS and DATS can induce cell cycle arrest and apoptosis in
many types of human cancer cell lines, but there is no information to
address DAS, DADS and DATS-affected drug resistance genes either in
vitro or in vivo studies. In the present study, we investigated the
effects of DAS, DADS and DATS on drug resistant gene expression in cold
205 cells. DAS, DADS and DATS also stimulated specific gene associated
with multi-drug resistance in colo 205 human colon cancer cells.

The human colon cancer cell line (colo 205) was obtained from the
Food Industry Research and Development Institute (Hsinchu, Taiwan). The
cells were maintained in RPM! 1640 medium supplemented with 10% FBS, 1%
penicillin-streptomycin (100 units/ml penicillin and 100 [micro]g/ml
streptomycin) and 2 mM L-glutamine in 75-[cm.sup.2] tissue culture
flasks and grown at 37 [degrees] C under a humidified 5% [CO.sub.2] and
95% air at one atmosphere.

In vitro studies

Real-time polymerase chain reaction (PCR)

It was used to examine effects of DAS, DADS and DATS on multi-drug
resistance genes in colo 205 cells. Cells (2 x [10.sup.6] cells/well) in
RPMI 1640 medium were plated in 12-well plates and allowed to grow for
24 h. The medium was replaced with fresh complete medium containing 25
[micro]M of DAS, DADS and DATS, respectively, for 24 and 48 h based on
our earlier studies (Lai et al. 2011; Yang et al. 2009). Stock solutions
of DAS, DADS and DATS were dissolved in DMSO, and an equal volume of
DMSO (final concentration 0.1%) was added to cells as a vehicle control.
At the end of the incubation period, cells were collected and suspended
in PBS by centrifugation. Total RNA was isolated using the Qiagen RNeasy
Mini Kit (Qiagen, Inc., Valencia, CA, USA) as previously described
(Chiang et al. 2011; Ji et al. 2009; Lu et al. 2010). RNA samples were
reverse-transcribed for 30 min at 42 [degrees] C with High Capacity cDNA
Reverse Transcription Kit according to the standard protocol of the
supplier (Applied Biosystems/Life Technologies, Carlsbad, CA, USA).
Quantitative PCR conditions were: 2 min at 50 [degrees] C, 10 min at 95
[degrees] C, and 40 cycles of 15 s at 95 [degrees] C, 1 min at 60
[degrees] C using 1 [micro]l of the cDNA reverse-transcribed as
described above, 2X SYBR Green PCR Master Mix (Applied Biosystems) and
200 nM of forward (F) and reverse (R) primers as shown in Table 1. Each
assay was run on an Applied Biosystems 7300 Real-Time PCR system
(Applied Biosystems/Life Technologies) in triplicate and expression
fold-changes were derived using the comparative CT method (Chiang et al.
2011; Lu et al. 2010).

The mice experiments were conducted according to institutional
guidelines approved by the Institutional Animal Care and Use Committee
(IACUC; No. 95-43-N), China Medical University (Taichung, Taiwan).
Thirty six-week-old male athymic nude mice were obtained from the
Laboratory Animal Center of National Applied Research Laboratories
(Taipei, Taiwan). Animals were maintained in standard vinyl cages with
air filter tops in a filtered laminar air flow room, food and water were
autoclaved and provided ad libitum. The outline of the experimental
design is shown in Fig. 3.

The colo 205 cells (1 x [10.sup.7] per mouse) were subcutaneously
(s.c.) injected into the flanks of mice as described previously (Ho et
al. 2009; Ji et al. 2009). Mice bearing tumors were randomly divided
into treatment groups (six mice per group). When xenografts reached
volumes of about 100 m[m.sup.3], the animals were then intraperitoneally
(i.p.) injected once every 4 days (in the morning) with 30 [micro]l of
control vehicle (DMSO), DAS or DADS or DATS (6 mg/kg), and doxorubicin
(8 mg/kg). Mice exhibiting tumors were monitored and counted, and the
tumor sizes were measured initially after 10 days for up to 32 days
after tumor cell inoculation. At the end of the study (4 weeks after
cell inoculation), animals were photographed and sacrificed. Tumors were
removed, measured and weighted. Total RNA from tumor tissues in each
group was collected for real-time PCR to examine multi-drug resistant
genes expression as described above.

Statistical analysis

Each value represents mean SD between the control and DAS, DADS and
DAIS-treated groups that were compared by one-way ANOVA followed by
Dunnett's test. * p < 0.05 and *** p < 0.001 were considered
significant.

Cells were treated with 25 p.M DAS, DADS and DAIS for 24 and 48 h
and gene expression levels in colo 205 cells were determined for MRP1,
MRP3, MRP4 and MRP6. Results presented in Fig. 2A-C indicated that DAS
and DADS enhanced Mdr3 gene expression at the 48-h treatment, but did
not affect MRP1, MRP4 and MRP6 gene expression in colo 205 cells (Fig.
2A and B). However, DAS and DADS inhibited the gene expressions of MRPs
at 24 and 48-h treatment, and both inhibited MRP3 at 24-h treatment and
inhibited MRP4 at 48 h treatment in colo 205 cells. Furthermore, DADS
treatment for 48 h inhibited MRP6 gene expressions. DATS treatment for
24 h led to significant inhibition of MRP3 gene expressions in colo 205
cells. It can be seen in Fig. 2C that DATS promoted MRP1 gene expression
at the 48 h treatment but did not affect MRP3, MRP4 and MRP6 gene
expression.

Fig. 5A and B show that DATS > DADS on increasing gene
expression of Mdr1 and MRP1. DAS did not significantly alter Mdr1 and
MRP1 gene expression levels. Data in Fig. 5C indicated that only DADS
stimulated MRP3 gene expression and showed that DADS and DATS promoted
MRP4 and MRP6 gene expression as seen in Fig. 5D and E.

Discussion

Cancer cells are able to develop resistance simultaneously to many
different anticancer drugs (Perez-Tomas 2006). Cancer cell resistance to
chemotherapy involves several mechanisms including mutation, drug
inactivation, over-expressions of the drug target genes or elimination
of the drug from the cell. Goldstein et al. showed that MDRl was
expressed in epithelial cancers derived from various organs/tissue
(colon, liver, kidney) and it was found in hematopoeitic cancers (AML,
ALL lymphoma) and solid tumors (breast and ovary cancer) (Goldstein et
al. 1989). The human MDR1 gene lies on chromosome 7 at q21.1 and its
polymorphisms have been reported in the MDR1 gene since 1989 (Callen et
al. 1987).

Many reports have shown that DAS, DADS and DATS inhibited
proliferation and apoptosis in different human cancer cell lines,
including colon cancer cell lines. However, the effects of DAS, DADS and
DATS on MDR gene expression in colon cancer cells have not been fully
reported. The previous report described by Demeule et al. indicated that
DADS has an effect of drug resistance and is able to increase
P-glycoprotein and promote the expression of multidrug
resistance-associated protein 2 (MRP-2) in vivo (Demeule et al. 2004).
In this study, we investigated and examined the effects of DAS, DADS and
DATS on MDRgene expression in human colon cancer cells (colo 205). DATS
promoted Mdrl gene expression in colo 205 cells, but DAS and DADS did
not affect Mdrl gene expression (Fig. 1). In in vivo studies, our
results also showed DADS and DATS promoted Mdrl gene expression but DATS
in xenograft colo 205 tumors after colo 205 cells were injected into
mice. Instead, DAS did not affect Mdrl gene expression in vivo (Fig.
5A).

The human MRP1 gene is mapped to chromosome 16pl3.1 (Slovak et al.
1993) and encompasses at least 200,000 base pairs containing 31 exons
(Grant et al. 1997). Cells overexpressing MRP1 protein are resistant to
a wide variety of anticancer drugs including doxorubicin (Chang et al.
1997), suggesting that they may be the substrates of MRP1 protein. In
the in vitro studies, we showed that colo 205 cells after exposure to
DAS, DADS and DATS for 24 and 48 h that DAS and DADS promoted Mdr3 gene
expression at the 48 h treatment but did not affect MRP1, MRP4 and MRP6
gene expression levels (Fig. 2A and B). DATS promoted MRP1 gene
expression at 48 h treatment but did not affect MRP3, MRP4 and MRP6 gene
expression. Several studies have shown that DAS, DADS and DATS induced
cytotoxicity in colon cancer cells and the order of effects was DAS <
DADS < DATS. However, the effects of these compounds on expression
levels of MDR genes are not exactly clear. Our results showed that DAS,
DADS and DATS presented different effects on drug-resistant gene
expression levels in colo 205 cells in vitro and in vivo. DATS has
greater stimulatory effects on drug resistance gene expression levels
but cytotoxicity in vitro and in vivo is higher for DATS than that of
DAS and DADS. Transcriptional activity may not be the primary mechanism
for MDR induced by DATS, DAS and DADS. Apparently, further
investigations are needed in the future.

Acknowledgement

This work was supported by the grant NSC 95-2320-B-039-030-MY2 from
National Science Council, Republic of China (Taiwan) and by the grant
CMUBH Rl 00-009 from China Medical University Beigang Hospital, Yunlin,
Taiwan R.O.C.